Aryl Sulfonic Pyridoxines as Antiplatelet Agents

ABSTRACT

Aryl sulfonic pyridoxine compounds with inhibition of serine protease activity and antiplatelet aggregation characteristics for the treatment of cardiovascular and cardiovascular related diseases are described. The methods are directed to administering pharmaceutical compositions comprising aryl sulfonic pyridoxines.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No. 10/974,707, filed Oct. 28, 2004, the entire disclosure of which is hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to aryl sulfonic pyridoxines and methods of treating cardiovascular, cerebrovascular, and cardiovascular related diseases or symptoms by administering pharmaceutical compositions comprising an aryl sulfonic pyridoxine.

BACKGROUND

Thrombosis, the development of blood clots within arterial vessels, is due to a complex mechanism involving the activation of both platelet aggregation and the coagulation protease cascade (Ann. Intern Med. (2001) 134: 224-38; N. Engl. J. Med. (2002) 347: 5-12; Thromb. Haemost. (2002) 86: 51-6). The pathways involved normally inhibit blood loss after vessel injury, but in thrombosis and related conditions, these reactions are inappropriately initiated and propagated.

On the molecular level, thrombosis is initiated by the release of mediators such as tissue factor (TF), von Willebrand Factor (vWF) (J. Thromb. Haemost. (2003) 1: 1602-1612), and collagen from ruptured atherosclerotic plaques or from damaged blood vessels. Collagen and vWF bind to receptors on platelets and initiate their activation. Once activated, platelets release secretory granules containing ADP, ATP, and calcium (Curr. Opin. Hematol. (2001) δ: 270-276). Activated platelets also synthesize and release thromboxane. The released ADP and thromboxane bind to receptors on the platelets to further propagate platelet activation. Once platelets are activated they start aggregating to initiate clot formation.

The trypsin-like family of serine proteases play an important role in thrombogenesis (Abbenante & Fairlie (2005) Med. Chem. 1: 71-104; Walenga et al., “Factor Xa Inhibitors” in Anticoagulants, Antiplatelets, and Thrombolytics (Mousa ed.), 93: 95-117 (2004), Humana Press Inc.). Tissue factor (TF), transiently exposed at a vascular site damaged either mechanically or by inflammatory processes, is responsible for initiating the coagulation cascade leading to thrombus formation (Giesen et al. (1999) Proc. Natl. Acad. Sci. USA 96: 2311-2315). Association of TF with activated factor VII (fvIIa) on the membrane surface triggers a complex enzyme amplification network of events that leads to the generation of thrombin and subsequently, conversion of fibrinogen to fibrin. In addition to its role in clot formation, thrombin amplifies its own production and activates platelets via its enzymatic action on platelet protease-activated receptors PAR-1 and PAR-4 (Lane (2005) Blood 106: 2605-2612).

Since the middle of the last century, anticoagulant therapy has consisted primarily of heparin treatment in acute situations, while vitamin k antagonists have been utilized for chronic therapy. Though these two regimens have proved indispensable in anticoagulant therapy, both approaches have shortcomings which have prompted the development of improved drugs in an attempt to address the limitations of these traditional agents. New approaches involve targeting specific pathways in the coagulation system in order to streamline anticoagulation (Weitz & Bates (2005) J. Thromb. Haemost. 3: 1843-1853). For example, factor Xa (fxa) in the prothrombinase complex generates thrombin (fIIa), and inhibiting either of these enzymes (Yavin et al. (2005) Eur. J. Intern. Med. 16: 257-266), or both Deng, et al. (2005) Bioorg. Med. Chem. Lett. 15: 4411-4416), has been the subject of recent drug design efforts. The notion of dual inhibition in the area of antithrombotics is not new, and in fact unfractionated heparin has several activities including the inhibition of the coagulation pathway serine proteases fIIa and fXa when complexed to antithrombin III.

Therefore, there is a need for compounds that inhibit the proteases of the blood and thus block platelet aggregation.

SUMMARY OF THE INVENTION

One embodiment of the invention includes aryl sulfonic pyridoxines, compositions containing the aryl sulfonic pyridoxines, and methods of treatment using therapeutically effective amounts of aryl sulfonic pyridoxines. Compounds and compositions of the invention can be used to treat cardiovascular, cerebovascular or related diseases and symptoms thereof.

The invention also provides an embodiment of the formula I:

wherein

R¹ is —OH, —O-alkyl, —(CH₂)_(n′) OH where n′ is an integer from 1 to 8, alkyl, cycloalkyl, or O-alkyl-aryl-R⁴, where R⁴ is —CN or amidine;

R² is alkyl; —(CH₂)_(n′)OH where n′ is as defined above; —(CH₂)_(n)COOH where n is an integer from 0 to 8; —(CH₂)_(n)COO(CH₂)_(n)CH₃ where n is as defined above; or R⁵ where R⁵ is (CH₂)_(n)-aryl-R⁶ where n is as defined above and R⁶ is SO₂NH₂ or SO₂NHC(CH₃)₃; (CH₂)_(n)-aryl-aryl-R⁶, where n and R⁶ are as defined above; (CH₂)_(n)—NH-aryl-aryl-R⁶, where n and R⁶ are as defined above; (CH₂)_(n)—NH—CO-aryl-aryl-R⁶, where n and R⁶ are as defined above; or —(CH₂)_(n)—NH-aryl-R⁶, where n and R⁶ are as defined above;

R³ is alkyl; —(CH₂)_(n′)OH where n′ is as defined above; or R⁵ where R⁵ is (CH₂)_(n)—NH-aryl-R⁶, where n and R⁶ are as defined above; (CH₂)_(n)—NH—CO-aryl-R⁶ where n and R⁶ are as defined above; (CH₂)_(n)—NH-aryl-aryl-R⁶ where n and R⁶ are as defined above; or (CH₂)_(n)—NH—CO-aryl-aryl-R⁶ where n and R⁶ are as defined above;

further wherein

-   -   R² is alkyl or —(CH₂)_(n′)OH where n′ is as defined above, and         R³ is R⁵; or     -   R² is R⁵ and R³ is alkyl or —(CH₂)_(n′)OH where n′ is as defined         above; and

R¹ and R² when taken together form compounds of formula II,

wherein R⁷ and R⁸ are independently H or CH₃, and R⁵ is as defined above; or a pharmaceutically acceptable salt thereof.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compounds of the formula I:

wherein

R¹ is —OH, —O-alkyl, —(CH₂)_(n′) OH where n′ is an integer from 1 to 8, alkyl, cycloalkyl, or O-alkyl-aryl-R⁴, where R⁴ is —CN or amidine;

R² is alkyl; —(CH₂)_(n′)OH where n′ is as defined above; —(CH₂)_(n′) COOH where n is an integer from 0 to 8; —(CH₂)_(n)COO(CH₂)_(n)CH₃ where n is as defined above; or R⁵ where R⁵ is (CH₂)_(n)-aryl-R⁶ where n is as defined above and R⁶ is SO₂NH₂ or SO₂NHC(CH₃)₃; (CH₂)_(n)-aryl-aryl-R⁶, where n and R⁶ are as defined above; (CH₂)_(n)—NH-aryl-aryl-R⁶, where n and R⁶ are as defined above; (CH₂)_(n)—NH—CO-aryl-aryl-R⁶, where n and R⁶ are as defined above; or —(CH₂)_(n)—NH-aryl-aryl-R⁶ where n and R⁶ are as defined above;

R³ is alkyl; —(CH₂)_(n′)OH where n′ is as defined above; or R⁵ where R⁵ is (CH₂)_(n)—NH-aryl-R⁶, where n and R⁶ are as defined above; (CH₂)_(n)—NH—CO-aryl-R⁶ where n and R⁶ are as defined above; (CH₂)_(n)—NH-aryl-aryl-R⁶ where n and R⁶ are as defined above; or (CH₂)_(n)—NH—CO-aryl-aryl-R⁶ where n and R⁶ are as defined above;

further wherein

-   -   R² is alkyl or —(CH₂)_(n′) OH where n′ is as defined above, and         R³ is R⁵; or     -   R² is R⁵ and R³ is alkyl or —(CH₂)_(n′) OH where n′ is as         defined above; and

R¹ and R² when taken together form compounds of formula II,

wherein R⁷ and R⁸ are independently H or CH₃, and R⁵ is as defined above;

or a pharmaceutically acceptable salt thereof.

The invention also provides an embodiment of the formula III:

wherein

R¹ is OH; OCH₃; OCH₂-(4-tert-Butyl-phenyl); or

where R⁴ is —CN or amidine;

R² is (CH₂)_(m)OH, where m=0 to 8; or R⁵, where R⁵ is

-   -    where W is (CH₂)_(n) where n=1, 2, or 3; where X is C=0 or         (CH₂)_(n′), where n′=0, 1, 2, or     -   3; Y is CH, CF, or N; and R⁶ is

R³ is (CH₂)_(m)OH, where m is as defined above; or R⁵, where R⁵ is as defined above;

further wherein

-   -   R² is (CH₂)_(m)OH, where m is as defined above, and R³ is R⁵; or     -   R² is R⁵ and R³ is (CH₂)_(m)OH, where m is as defined above; and

R¹ and R² when taken together form a compound of formula IV

wherein R⁷ and R⁸ are independently H or CH₃; and R⁵ is as defined above;

or a pharmaceutically acceptable salt thereof.

As used herein “alkyl” includes a saturated linear or branched hydrocarbon radical. In one embodiment, alkyl has from 1 to 8 carbon atoms. In another embodiment, alkyl has from 1 to 6 carbon atoms. In another embodiment, alkyl has from 1 to 4 carbon atoms. In one embodiment, alkyl has 1 carbon. The alkyl group may optionally be substituted with one or more substituents such as fluorine, chlorine, alkoxy groups having from 1 to 8 carbon atoms (e.g., methoxy or ethoxy), or amido groups having from 1 to 8 carbon atoms, such as acetamido. These substituents may themselves be substituted with one or more functional groups such as hydroxy groups, carboxy groups, acetoxy groups, or halogens.

As used herein “cycloalkyl” refers to a cyclic hydrocarbon having from 3 to 8 carbon atoms, preferably 3 to 6 carbon atoms, such as, for example, cyclopropyl, cyclopentyl, cyclohexyl, and the like.

As used herein “aryl” means a mono- or poly-nuclear aromatic hydrocarbon radical. Examples of “aryl” groups include, but are not limited to aromatic hydrocarbons such as a phenyl group or a naphthyl group. The aromatic group may optionally be substituted with one or more substituents such as fluorine, chlorine, alkyl groups having from 1 to 10 carbon atoms (e.g., methyl or ethyl), alkoxy groups having from 1 to 8 carbon atoms (e.g., methoxy or ethoxy), alkoxyalkyl groups having from 1 to 8 carbon atoms and one or more oxygen atoms, or amido groups having from 1 to 8 carbon atoms, such as acetamido. These substituents may themselves be substituted with one or more functional groups such as hydroxy groups, carboxy groups, acetoxy groups, or halogens.

In one embodiment, aryl is a phenyl group or a naphthyl group that is either unsubstituted or substituted.

In another embodiment, aryl is a heteroaryl in which one or more of the carbon atoms of an aromatic hydrocarbon is substituted with a nitrogen, sulfur, or oxygen. Examples of a “heteroaryl” include, but are not limited to pyridine, pyrimidine, pyran, dioxin, oxazine, and oxathiazine. Likewise, the heteroaryl may optionally be substituted with functional groups such as hydroxy groups, carboxy groups, halogens, and amino groups.

As used herein, “amidine” means a group having the formula

The invention also includes pharmaceutically acceptable salts of the compounds of the invention. The compounds of the invention are capable of forming both pharmaceutically acceptable acid addition and/or base salts. Pharmaceutically acceptable acid addition salts of the compounds of the invention include salts derived from nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydriodic, hydrofluoric, phosphorous, and the like, as well as the salts derived from nontoxic organic acids, such as aliphatic mono- and di-carboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Such salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, trifluoroacetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, and the like. Also contemplated are salts of amino acids such as arginate and the like and gluconate, galacturonate, n-methyl glucamine, etc. (see Berge et al., J. Pharmaceutical Science, 66: 1-19 (1977). The term “pharmaceutically acceptable salts” also includes any pharmaceutically acceptable base salt including, but not limited to, amine salts, trialkyl amine salts and the like. Such salts can be formed quite readily by those skilled in the art using standard techniques.

The acid addition salts of the basic compounds are prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt in the conventional manner. The free base form may be regenerated by contacting the salt form with a base and isolating the free base in the conventional manner. The free base forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free base for purposes of the present invention. Base salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations include, but are not limited to, sodium, potassium, magnesium, and calcium. Examples of suitable amines are N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, and procaine.

Some of the compounds described herein contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms which may be defined in terms of absolute stereochemistry as (R)- or (S)- . The present invention is meant to include all such possible diastereomers and enantiomers as well as their racemic and optically pure forms. Optically active (R)- and (S)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise all tautomeric forms are intended to be included.

General Methods of Preparing Compounds of Formulae I, II, III, and IV

The compounds are generally prepared by combining an aldehyde or a carboxylate with an amine group to produce an elaborated pyridine structure. The general scheme of preparing the compounds of the formulae comprise protecting the hydroxyl groups at R₁ and R₂ of pyridoxine with known blocking groups such as esters, ethers, cyclic acetals, cyclic ketals, etc. and elaborating R³ through generating an aldehyde, acid, halide, or amine functionality as shown in Schemes 1-4. R³ may be an aryl or biaryl containing a nitro, amino, or cyano group that can be converted to an amidine by known chemical procedures. Additionally, protecting R¹ and R³ with known blocking groups such as esters, ethers, cyclic acetals, cyclic ketals, etc. and elaborating R² through generating an aldehyde, acid, halide, or amine functionality can be achieved through the same general scheme as shown in Scheme 5.

where the dashed lines are (CH₂)_(n) where n=0-8.

where the dashed lines are (CH₂)_(n) and n=0-8.

where the dashed lines are (CH₂)_(n) and n=0-8.

where the dashed lines are (CH₂)_(n) and n=0-8.

where R³ is (CH₂)_(n)—Ar—X, where n=0-8 and Ar—X is any aromatic terminating in —SO₂NH₂ or —SO₂NH₂C(CH₃)₃.

Other positions on the pyridoxine ring can also be substituted according to the aforementioned general scheme. Substitutions are not specific to the positions described above.

Conditions to Be Treated

In one embodiment of the invention, compounds of the invention can be used to treat cardiovascular or related diseases. Cardiovascular or related diseases include, for example, cerebral ischemia, cerebral hemorrhage, ischemic stroke, hemorrhagic stroke, hypertension, myocardial infarction, ischemia reperfusion injury, myocardial ischemia, congestive heart failure, blood coagulation disorders, cardiac hypertrophy, and platelet aggregation. Cardiovascular or related diseases also include diseases that arise from thrombotic and prothrombotic states in which the coagulation cascade is activated such as, for example, deep vein thrombosis, disseminated intravascular coagulopathy, and pulmonary embolism.

Heart failure is a pathophysiological condition in which the heart is unable to pump blood at a rate commensurate with the requirement of the metabolizing tissues or can do so only from an elevated filling pressure (increased load). Thus, the heart has a diminished ability to keep up with its workload. Over time, this condition leads to excess fluid accumulation, such as peripheral edema, and is referred to as congestive heart failure.

When an excessive pressure or volume load is imposed on a ventricle, myocardial hypertrophy (i.e., enlargement of the heart muscle) develops as a compensatory mechanism. Hypertrophy permits the ventricle to sustain an increased load because the heart muscle can contract with greater force. However, a ventricle subjected to an abnormally elevated load for a prolonged period eventually fails to sustain an increased load despite the presence of ventricular hypertrophy, and pump failure can ultimately occur.

Heart failure can arise from any disease that affects the heart and interferes with circulation. For example, a disease that increases the heart muscle's workload, such as hypertension, will eventually weaken the force of the heart's contraction. Hypertension is a condition in which there is an increase in resistance to blood flow through the vascular system. This resistance leads to increases in systolic pressure, diastolic blood pressure, or both. Hypertension places increased tension on the left ventricular myocardium, causing it to stiffen and hypertrophy, and accelerates the development of atherosclerosis in the coronary arteries. The combination of increased demand and lessened supply increases the likelihood of myocardial ischemia leading to myocardial infarction, sudden death, arrhythmias, and congestive heart failure.

Ischemia is a condition in which an organ or a part of the body fails to receive a sufficient blood supply. When an organ is deprived of a blood supply, it is said to be hypoxic. An organ will become hypoxic even when the blood supply temporarily ceases, such as during a surgical procedure or during temporary artery blockage. Ischemia initially leads to a decrease in or loss of contractile activity. When the organ effected is the heart, this condition is known as myocardial ischemia, and myocardial ischemia initially leads to abnormal electrical activity. This can generate an arrhythmia. When myocardial ischemia is of sufficient severity and duration, cell injury can progress to cell death—i.e., myocardial infarction—and subsequently to heart failure, hypertrophy, or congestive heart failure.

Ischemic reperfusion of the organ occurs when blood flow resumes to an organ after temporary cessation. For example, reperfusion of an ischemic myocardium can counter the effects of coronary occlusion, a condition that leads to myocardial ischemia. Ischemic reperfusion to the myocardium can lead to reperfusion arrhythmia or reperfusion injury. The severity of reperfusion injury is affected by numerous factors, such as, for example, duration of ischemia, severity of ischemia, and speed of reperfusion. Conditions observed with ischemia reperfusion injury include neutrophil infiltration, necrosis, and apoptosis.

Pharmaceutical Compositions

Although it is possible for compounds of the invention to be administered alone in a unit dosage form, the compounds are typically administered in admixture with a carrier as a pharmaceutical composition to provide a unit dosage form. The invention provides pharmaceutical compositions containing at least one compound of the invention. A pharmaceutical composition comprises a pharmaceutically acceptable carrier in combination with a compound of the invention or a pharmaceutically acceptable salt of a compound of the invention.

A pharmaceutically acceptable carrier includes, but is not limited to, physiological saline, ringers, phosphate-buffered saline, and other carriers known in the art. Pharmaceutical compositions can also include additives such as, for example, stabilizers, antioxidants, colorants, excipients, binders, thickeners, dispersing agents, readsorpotion enhancers, buffers, surfactants, preservatives, emulsifiers, isotonizing agents, and diluents. Pharmaceutically acceptable carriers and additives are chosen such that side effects from the pharmaceutical compound are minimized and the performance of the compound is not canceled or inhibited to such an extent that treatment is ineffective.

Methods of preparing pharmaceutical compositions containing a pharmaceutically acceptable carrier in combination with a therapeutic compound of the invention or a pharmaceutically acceptable acid addition salt of a compound of the invention are known to those of skill in the art. All methods can include the step of bringing the compound of the invention in association with the carrier and additives. The formulations generally are prepared by uniformly and intimately bringing the compound of the invention into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired unit dosage forms.

For oral administration as a tablet or capsule, the compositions can be prepared according to techniques well known in the art of pharmaceutical formulation. The compositions can contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavoring agents. As immediate release tablets, the compositions can contain microcrystalline cellulose, starch, magnesium stearate and lactose or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.

For administration by inhalation or aerosol, the compositions can be prepared according to techniques well known in the art of pharmaceutical formulation. The compositions can be prepared as solutions in saline, using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons or other solubilizing or dispersing agents known in the art.

For administration as injectable solutions or suspensions, the compositions can be formulated according to techniques well-known in the art, using suitable dispersing or wetting and suspending agents, such as sterile oils, including synthetic mono- or di-glycerides, and fatty acids, including oleic acid.

For rectal administration as suppositories, the compositions can be prepared by mixing with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ambient temperatures, but liquefy or dissolve in the rectal cavity to release the drug.

Method of Treatment Using Compounds of the Invention

In another aspect of the invention, methods are provided for the treatment of cardiovascular or related diseases and symptoms thereof.

As used herein, the terms “treatment” and “treating” include inhibiting, alleviating, and healing cardiovascular or related diseases or symptoms thereof. Treatment can be carried out by administering a therapeutically effective amount of at least one compound of the invention. A “therapeutically effective amount” as used herein includes a prophylactic amount, for example an amount effective for alleviating or healing the above mentioned diseases or symptoms thereof.

A physician or veterinarian of ordinary skill readily determines a mammalian subject who is exhibiting symptoms of any one or more of the diseases described above. Regardless of the route of administration selected, a compound of the invention or a pharmaceutically acceptable acid addition salt of a compound of the invention can be formulated into pharmaceutically acceptable unit dosage forms by conventional methods known in the pharmaceutical art. An effective but nontoxic quantity of the compound is employed in treatment. The compounds can be administered in enteral unit dosage forms, such as, for example, tablets, sustained-release tablets, enteric coated tablets, capsules, sustained-release capsules, enteric coated capsules, pills, powders, granules, solutions, and the like. They can also be administered parenterally, such as, for example, subcutaneously, intramuscularly, intradermally, intramammarally, intravenously, and by other administrative methods known in the art.

The ordinarily skilled physician or veterinarian will readily determine and prescribe the therapeutically effective amount of the compound to treat the disease for which treatment is administered. In so proceeding, the physician or veterinarian could employ relatively low dosages at first, subsequently increasing the dose until a maximum response is obtained. Typically, the particular disease, the severity of the disease, the compound to be administered, the route of administration, and the characteristics of the mammal to be treated, for example, age, sex, and weight, are considered in determining the effective amount to administer. Administering a therapeutic amount of a compound of the invention for treating cardiovascular or related diseases or symptoms thereof, is in a range of about 0.1-100 mg/kg of a patient's body weight, more preferably in the range of about 0.5-50 mg/kg of a patient's body weight, per daily dose. The compound can be administered for periods of short and long duration. Although some individual situations can warrant to the contrary, short-term administration, for example, 30 days or less, of doses larger than 25 mg/kg of a patient's body weight is preferred to long-term administration. When long-term administration, for example, months or years, is required, the suggested dose usually does not exceed 25 mg/kg of a patient's body weight.

A therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable addition salt of a compound of the invention for treating the above-identified diseases or symptoms thereof can be administered prior to, concurrently with, or after the onset of the disease or symptom. A compound of the invention can be administered concurrently. “Concurrent administration” and “concurrently administering” as used herein includes administering a compound of the invention and another therapeutic agent in admixture, such as, for example, in a pharmaceutical composition or in solution, or separately, such as, for example, separate pharmaceutical compositions or solutions administered consecutively, simultaneously, or at different times but not so distant in time such that the compound of the invention and the other therapeutic agent cannot interact and a lower dosage amount of the active ingredient cannot be administered.

In one embodiment of the invention, a method is provided for treating cardiovascular or related diseases comprising administering to a mammal a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable addition salt of a compound of the invention in a unit dosage form. The cardiovascular or related diseases that can be treated include hypertrophy, hypertension, congestive heart failure, heart failure subsequent to myocardial infarction, myocardial ischemia, cerebral ischemia, ischemia reperfusion injury, arrhythmia, myocardial infarction, blood coagulation, platelet aggregation, atherosclerosis, atrial fibrillation, or acute coronary syndrome. Preferably, the cardiovascular disease treated is hypertrophy, congestive heart failure, arrhythmia, or ischemia reperfusion injury. Compounds of the invention may also be administered as anticoagulants to patients with heparin induced thrombocyotpenia (HIT).

Serine protease activity can be inhibited by the administration of a compound of the invention or a pharmaceutically acceptable acid addition salt of a compound of the invention. Compounds of the invention can be administered to inhibit serine protease activity in a mammal, thereby affecting varying disease states, including platelet aggregation. Compounds of the invention can inhibit serine proteases such as, but not limited to, trypsin, chymotrypsin, kallikrein, elastase, thrombin (Factor IIa), plasmin, Factors VIIa, IXa, Xa, XIa, XIIa, and protein C. Preferably, the mammal is a human. Compounds of the invention can also be used to inhibit serine protease activity in vitro. Serine protease inhibitors can be useful for research and diagnostic purposes. For instance, compounds of the invention can inhibit serine protease activity in a monolayer of a cell culture following contact with the cells of said cell culture.

In another embodiment, compounds of the invention can be administered to inhibit non-mammalian serine proteases. The non-mammalian serine protease can be from microorganisms, including bacteria and viruses. Bacterial serine proteases include, but is not limited to, subtilisin (from Bacillus subtilis). Viral serine proteases can be inhibitied by compounds disclosed herein, including, but not limited to, hepatitis C virus (HCV) serine proteases, for example the HCV serine protease NS3. Additionally, compounds disclosed herein can be used to treat viral infections. Treating viral infections can include administering compounds disclosed herein in order to inhibit viral serine proteases (e.g., HCV) or to inhibit mammalian serine proteases necessary for viral infection (e.g., human immunodeficiency virus (HIV); see, e.g., U.S. Pat. No. 6,849,605).

The compound of the invention can also be administered to treat cardiovascular diseases and other diseases that arise from thrombotic and prothrombotic states in which the coagulation cascade is activated, such as, for example, deep vein thrombosis, disseminated intravascular coagulopathy, Kasabach-Merritt syndrome, pulmonary embolism, myocardial infarction, stroke, thromboembolic complications of surgery, and peripheral arterial occlusion. A compound of the invention may also be useful in the treatment of adult respiratory distress syndrome, septic shock, septicemia, or inflammatory responses, such as edema and acute or chronic atherosclerosis, because thrombin has been shown to activate a large number of cells outside of the coagulation process, such as, for example, neutrophils, fibroblasts, endothelial cells, and smooth muscle cells.

The method for treating cardiovascular or related diseases can further comprise concurrent administration of other therapeutic agents already known to be suitable for treating the above-identified diseases. For example, methods of the invention include concurrently administering a compound of the invention or a pharmaceutically acceptable acid addition salt of a compound of the invention in combination with a therapeutic cardiovascular compound to treat hypertrophy, hypertension, congestive heart failure, heart failure subsequent to myocardial infarction, myocardial ischemia, ischemia reperfusion injury, arrhythmia, or myocardial infarction. Preferably, the cardiovascular disease treated is hypertrophy, congestive heart failure, arrhythmia, or ischemia reperfusion injury.

The compounds of the invention can also be used in combination with other therapeutic cardiovascular compounds that are generally used to treat cardiovascular or related diseases as well as symptoms thereof. A skilled physician or veterinarian readily determines a subject who is exhibiting symptoms of any one or more of the diseases described above and makes the determination about which compound is generally suitable for treating specific cardiovascular conditions and symptoms.

For example, myocardial ischemia can be treated by the administration of a compound of the invention or a pharmaceutically acceptable acid addition salt of a compound of the invention concurrently with another therapeutic agent. Other suitable therapeutic agents include, for example, a angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist, a calcium channel blocker, an antithrombolytic agent, a β-adrenergic receptor antagonist, a diuretic, an α-adrenergic receptor antagonist, or a mixture thereof.

As another example, congestive heart failure can be treated by the administration of a compound of the invention or a pharmaceutically acceptable acid addition salt of a compound of the invention concurrently with another therapeutic agent. Other suitable therapeutic agents include, for example, an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist, a calcium channel blocker, a vasodilator, a diuretic, or a mixture thereof.

Myocardial infarction can be treated by the administration of a compound of the invention or a pharmaceutically acceptable acid addition salt of a compound of the invention concurrently with another therapeutic agent. Other suitable therapeutic agents include, for example, an angiotensin converting enzyme inhibitor, a calcium channel blocker, an antithrombolytic agent, a β-adrenergic receptor antagonist, a diuretic, an α-adrenergic receptor antagonist, or a mixture thereof.

Hypertension can be treated by the administration of a compound of the invention or a pharmaceutically acceptable acid addition salt of a compound of the invention concurrently with another therapeutic agent. Other suitable therapeutic agents include, for example, an angiotensin converting enzyme inhibitor, a calcium channel blocker, a β-adrenergic receptor antagonist, a vasodilator, a diuretic, an α-adrenergic receptor antagonist, or a mixture thereof.

Arrhythmia can be treated by the administration of a compound of the invention or a pharmaceutically acceptable acid addition salt of a compound of the invention concurrently with another therapeutic agent. Other suitable therapeutic agents include, for example, a calcium channel blocker, a β-adrenergic receptor antagonist, or a mixture thereof.

Blood clots in the arteries (arterial thrombosis) or veins (venous thrombosis) can be reduced or removed by the administration of a compound of the invention or a pharmaceutically acceptable acid addition salt of a compound of the invention concurrently with an anti-platelet agent such as clopidogrel (aspirin, dipyridamole, etc.), glycoprotein IIb/IIIa inhibitor such as eptifibatide (INTEGRILIN®), or by an anticoagulant such as UFH (unfractionated heparins), LMWH (low molecular weight heparins), hirudin, or argatroban.

Hypertrophy can be treated by the administration of a compound of the invention or a pharmaceutically acceptable acid addition salt of a compound of the invention concurrently with another therapeutic agent. Other suitable therapeutic agents include, for example, an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist, a calcium channel blocker, or a mixture thereof.

Ischemia reperfusion injury can be treated by the administration of a compound of the invention or a pharmaceutically acceptable acid addition salt of a compound of the invention concurrently with another therapeutic agent. Other suitable therapeutic agents include, for example, an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist, a calcium channel blocker, or a mixture thereof.

Compounds of the invention or pharmaceutically acceptable salts thereof can be administered post-surgically, alone or concurrently with other suitable therapeutic agents. For instance, the method would include, but is not limited to, administration to patients following hip replacement surgery, or invasive cardiovascular surgery, including coronary artery bypass graft (CABG), endarectomy, and heart valve replacement. Compounds of the invention or pharmaceutically acceptable salts thereof can be administered, alone or concurrently with other suitable therapeutic agents, following any angioplasty procedure. For instance, administration of said compounds may follow percutaneous transluminal angioplasty (PTA). PTA is used in coronary, pulmonary, peripheral, intracranial, extracranial carotid, renal, and aortic stenoses.

Additionally, medical devices can be coated with the compounds of the invention or pharmaceutically acceptable acid salts of the compound alone or in mixture with other suitable therapeutic agents (e.g., an angiotensin converting enzyme inhibitor). Medical devices that can be coated with the compounds of the invention or pharmaceutically acceptable salts thereof alone or in mixture with other suitable therapeutic agents include, but are not limited to, intravascular stents and catheters. Intravascular stents are used to prevent blood vessel wall collapse. Drug-eluting stents are coated with a mixture of polymers and drug to prevent restenosis. Examples of drug-eluting stents are the CYPHER™ sirolimus-eluting stent (Cordis Corp., Miami, Fla.) and TAXUS™ paclitaxel-eluting stent (Boston Scientific Corp., Natick, Mass.).

This invention is further characterized by the following examples. These examples are not meant to limit the scope of the invention but are provided for exemplary purposes to more fully describe the invention. Variation within the scope of the invention will be apparent to those skilled in the art.

EXAMPLES

All reagents used were purchased from standard commercial sources, or synthesized by known literature methods. HPLC analysis was performed using a Waters 996 PDA high performance liquid chromatograph equipped with a Waters 600 controller. Signals were detected with a photodiode array detector (set at max plot 254-400 nm). NMR spectra were recorded on a Bruker AM-300 instrument (¹³C, ¹⁹F and ³¹P at 75.5, 282 and 121 MHz respectively) and were calibrated using residual nondeuterated solvent as the internal reference. All ¹⁹F spectra are reported using hexafluorobenzene (δ −162.9 ppm) as the external standard while ³¹p spectra were collected using 85% H₃PO₄ (δ0.0 ppm) as the external reference.

Example 1 Synthesis of 4-[(2,2,8-Trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-ylmethyl)-amino]-biphenyl-2-sulfonic acid tert-butylamide (1)

To a 250 mL three neck flask fitted with a condenser and Dean-Stark apparatus was added 4′-amino-biphenyl-2-sulfonic acid tert-butylamide (1.22 g, 4.0 mmol), p-toluenesulfonic acid monohydrate (152 mg, 0.8 mmol), 2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridine-5-carbaldehyde (995 mg, 4.8 mmol, see Korytnyk et al., Methods Enzymol. 1970; 18A: 524-566) and toluene (120 ml). The reaction mixture was stirred at 120° C. under nitrogen atmosphere for 7 h before concentrating to dryness. The resulting solid was then dissolved in acetic acid (20 mL), cooled to 0° C., and sodium borohydride (529 mg, 14 mmol) was added slowly. After the addition of sodium borohydride was complete, dichloromethane (30 mL) was added to the reaction mixture and stirring was continued at room temperature for an additional 3 h. Sodium hydroxide (5 N) was added to neutralize the solution, and the reaction mixture was extracted with ethyl acetate, dried over MgSO₄, filtered and evaporated. The crude mixture was purified by column chromatography on silica gel using a mixture of ethyl acetate and hexane (1:1) as eluant, to give 4′-[(2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-ylmethyl)-amino]-biphenyl-2-sulfonic acid tert-butylamide (1) (0.457 g, 24% yield) as a colorless solid.

¹H-NMR (CDCl₃): δ 8.13 (d, 1H), 8.04 (s, 1H), 7.52 (t, 1H), 7.41 (t, 1H), 7.35 (d, 2H), 7.28 (d, 1H), 6.70 (d, 2H), 4.90 (s, 2H), 4.20 (d, 2H), 3.99 (t, 1H), 3.69 (s, 1H), 2.41(s, 3H), 1.56 (s, 6H), 0.98 (s, 9H).

Example 2 Synthesis of 4′-[(5-Hydroxy-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl) -amino]-biphenyl-2-sulfonic acid tert-butylamide (2)

To a solution of 10% formic acid in water (50 mL) was added 3-[(2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-ylmethyl)-amino]-benzonitrile (1) (336 mg, 0.7 mmol) and the reaction mixture was heated at 100° C. under nitrogen atmosphere. The reaction mixture was then concentrated to dryness. The resulting pale yellow solid was dissolved in small amount of dichloromethane and diethyl ether was added to induce precipitation of a yellow solid. The pale yellow 4′-[(5-hydroxy-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl)-amino]-biphenyl-2-sulfonic acid tert-butylamide (2) (215 mg, 70% yield) was collected by filtration.

¹H-NMR (DMSO-d6): δ 8.70 (s, 1H), 8.07 (d, 1H), 7.77 (s, 1H), 7.60 (m, 2H), 7.43 (s, 4H), 7.33 (d, 1H), 5.14 (s, 2H), 4.77 (s, 2H), 2.33 (s, 3H), 0.98 (s, 9H).

Example 3 Synthesis of 4′-[(5-Hydroxy-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl)-amino]-biphenyl-2-sulfonic acid amide (3)

Hydrogen chloride gas was bubbled into a suspension of 4′-[(5-hydroxy-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl)-amino]-biphenyl-2-sulfonic acid tert-butylamide (2) (160 mg, 0.36 mmol) in methyl alcohol (20 mL) at 0° C. for 10 min. The solvent was evaporated and the products were purified on a silica gel column using a mixture of methyl alcohol:dichloromethane (1:9) as eluant to give 4′-[(5-hydroxy-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl)-amino]-biphenyl-2-sulfonic acid amide (3) (139 mg, 25% yield).

¹H-NMR (CD₃OD): δ 8.08 (d, 1H), 7.92 (s, 1H), 7.59 (t, 1H), 7.47 (t, 1H), 7.33 (d, 1H), 7.24 (d, 2H), 6.74 (d, 2H), 4.99 (s, 2H), 4.36 (s, 2H), 2.43 (s, 3H). MS m/z (ES⁺): 400.22 (M+H⁺).

Example 4 Synthesis of 2′-tert-Butylsulfamoyl-biphenyl-4-carboxylic acid (2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-ylmethyl)-amide (4)

A mixture of 2′-tert-butylsulfamoyl-biphenyl-4-carboxylic acid mono-sodium salt (200 mg, 0.56 mmol), 2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-yl)-methylamine (123 mg, 0.59 mmol), 1-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (EDC) (226 mg, 1.18 mmol), and 4-(dimethylamino)pyridine (DMAP) (144 mg, 1.18 mmol) in anhydrous dichloromethane (25 mL) was stirred at room temperature under nitrogen atmosphere overnight. The reaction mixture was concentrated and the crude mixture was purified by column chromatography on silica gel using a mixture of methyl alcohol:dichloromethane (1:9) as eluant to give 2′-tert-butylsulfamoyl-biphenyl-4-carboxylic acid (2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-ylmethyl)-amide (4) (196 mg, 67% yield) as a colorless solid.

¹H-NMR (CDCl₃): δ 8.16 (s, 1H), 8.04 (d, 1H), 7.94 (d, 2H), 7.57-7.46 (m, 5H), 5.01(s, 2H), 4.58 (d, 2H), 4.03 (s, 1H), 2.50 (s, 3H), 1.56 (s, 6H), 1.01 (s, 9H).

Example 5 Synthesis of 2′-tert-Butylsulfamoyl-biphenyl-4-carboxylic acid (5-hydroxy-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl)-amide (5)

The hydrolysis of 2′-tert-butylsulfamoyl-biphenyl-4-carboxylic acid (2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-ylmethyl)-amide (4) (300 mg, 0.57 mmol), following the procedure described in Example 2, gave 2′-tert-butylsulfamoyl-biphenyl-4-carboxylic acid (5-hydroxy-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl)-amide (5) (219 mg, 79% yield) as a colorless solid.

¹H-NMR (CD₃OD): δ 8.32 (m, 4H), 8.16 (s, 1H), 8.09 (d, 2H), 7.84 (t, 1H), 7.75 (d, 3H), 7.54 (d, 1H), 5.23 (s, 2H), 4.84 (s, 2H), 2.66 (s, 3H), 1.25 (s, 9H). MS m/z (ES⁺): 484.41 (M+H⁺).

Example 6 Synthesis of 2′-Sulfamoyl-biphenyl-4-carboxylic acid (5-hydroxy-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl)-amide (6)

The hydrolysis of 2′-tert-butylsulfamoyl-biphenyl-4-carboxylic acid (5-hydroxy-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl)-amide (5) (101 mg, 0.21 mmol), following the procedure described in Example 3, gave 2′-sulfamoyl-biphenyl-4-carboxylic acid (5-hydroxy-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl)-amide (6) (219 mg, 79% yield) as a colorless solid.

¹H-NMR (CD₃OD): δ 8.27 (m, 2H), 8.05 (d, 2H), 7.81-7.72 (m, 2H), 7.68 (d, 2H), 7.48 (d, 1H), 5.35 (s, 2H), 4.85 (s, 2H), 2.78 (s, 3H). MS m/z (ES⁺): 428.29 (M+H⁺).

Example 7 Synthesis of 4′-[(3-Hydroxy-5-hydroxymethyl-2-methyl-pyridin-4-ylmethyl)-amino]-biphenyl-2-sulfonic acid tert-butylamide (7)

A mixture of pyridoxal hydrochloride (330 mg, 1.62 mmol), 4′-amino-biphenyl-2-sulfonic acid tert-butylamide (494 mg, 1.62 mmol), p-toluenesulfonic acid monohydrate (68 mg, 0.36 mmol) and toluene (150 mL) was added to a 250 mL three-necked round bottom flask fitted with a condenser and a Dean-Stark trap, and heated at 100° C. under nitrogen atmosphere for 3 h. The solvent was then evaporated and the crude product was dissolved in dichloromethane (70 mL), cooled down to 0° C. and then sodium borohydride (163 mg, 4.32 mmol) and methyl alcohol (15 mL) were added. The reaction mixture was stirred at room temperature overnight, after which time the solvent was removed. The residue was diluted with saturated aqueous sodium bicarbonate and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, filtered and evaporated. The crude product was purified by column chromatography on silica gel using a mixture of dichloromethane:methyl alcohol (9:1) as eluant to give 4′-[(3-hydroxy-5-hydroxymethyl-2-methyl-pyridin-4-ylmethyl)-amino]-biphenyl-2-sulfonic acid tert-butylamide (7) (178 mg, 24% overall yield for two steps) as a colorless solid.

¹H-NMR (CDCl₃): δ 8.13 (dd, 1H), 7.84 (s, 1H), 7.53 (td, 1H), 7.44 (td, 1H), 7.37 (d, 2H), 7.27 (dd, 1H), 6.89 (d, 2H), 4.68 (s, 2H), 4.61 (s, 2H), 3.71 (s, 1H), 2.44 (s, 3H), 0.98 (s, 9H). MS m/z (ES⁺): 456.29 (M+H⁺).

Example 8 Synthesis of 4′-[(3-Hydroxy-5-hydroxymethyl-pyridin-4-ylmethyl)-amino]-biphenyl-2-sulfonic acid amide (8)

The hydrolysis of 4′-[(3-hydroxy-5-hydroxymethyl-2-methyl-pyridin-4-ylmethyl)-amino]-biphenyl-2-sulfonic acid tert-butylamide (7) (75 mg, 0.16 mmol), following the procedure described in Example 3, gave 4′-[(3-hydroxy-5-hydroxymethyl-pyridin-4-ylmethyl)-amino]-biphenyl-2-sulfonic acid amide (8) (49 mg, 76% yield) as a colorless solid.

¹H-NMR (CD₃OD): δ 8.09 (s, 1H), 7.92 (dd, 1H), 7.45-7.38 (m, 2H), 7.28 (dt, 2H), 7.16-7.10 (m, 3H), 4.70 (s, 2H), 4.66 (s, 2H), 2.53 (s, 3H). MS m/z (ES⁺): 400.28 (M+H⁺).

Example 9 Synthesis of 3′-Fluoro-4′-[(3-hydroxy-5-hydroxymethyl-2-methyl-pyridin-4-ylmethyl)-amino]-biphenyl-2-sulfonic acid tert-butylamide (9)

The reductive amination of pyridoxal hydrochloride (436.4 mg, 2.143 mmol) and 4′-amino-3′-fluoro-biphenyl-2-sulfonic acid tert-butylamide (760 mg, 2.357 mmol), following the procedure described in Example 7, gave 3′-fluoro-4′-[(3-hydroxy-5-hydroxymethyl-2-methyl-pyridin-4-ylmethyl)-amino]-biphenyl-2-sulfonic acid tert-butylamide (9).

¹H-NMR (CD₃OD): δ 8.06 (d, 1H), 7.9 (br s, 1H), 7.57 (t, 1H), 7.47 (t, 1H), 7.3 (d, 1H), 7.1 (dd, 1H), 7.07 (d, 1H), 7.01 (t, 1H), 4.75 (s, 2H), 4.61 (s, 2H), 0.98 (s, 9H). ⁹F-NMR (CD₃OD; ¹H-decoupled): δ −135.5.

Example 10 Synthesis of (5-Bromo-pyridin-2-yl)-(2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridine-5-ylmethyl)-amine (10)

Compound (10) was prepared according to the procedure described in Example 1 from 2-amino-5-bromopyridine (4.33 g, 25 mmol) and 2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridine-5-carbaldehyde (5.18 g, 25 mmol). The crude mixture was purified by column chromatography over silica gel using a hexane/ethyl acetate (1:1) mixture as eluant to furnish 1.8 g (20%) of (10).

¹H-NMR CD₃OD): δ 7.99 (d, 1H), 7.89 (s, 1H), 7.5 (dd, 1H), 6.5 (d, 1H), 4.95 (s, 2H), 4.37 (s, 2H), 2.33 (s, 3H), 1.56 (s, 6H).

Example 11 Synthesis of N-tert-Butyl-2-[6-(2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-ylamino)-pyridin-3-yl]-benzenesulfonamide (11)

A mixture of (5-bromo-pyridin-2-yl)-(2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-yl)-amine (10) (564 mg, 1.55 mmol), tetrakis(triphenylphosphine) palladium(0) (174 mg, 0.15 mmol), cesium carbonate (1.56 g, 4.8 mmol), 2-tert-butylsulfamoyl-phenylboronic acid (438 mg, 1.7 mmol) in a solution of toluene (20 mL), iso-butyl alcohol (15 mL) and water (5 mL) was stirred at 80° C. under nitrogen for 5 h. The reaction was diluted with water and extracted with ethyl ether. The organic layer was dried over anhydrous magnesium sulfate, filtered and evaporated. The crude product was purified by column chromatography on silica gel to give N-tert-butyl-2-[6-(2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-ylamino)-pyridin-3-yl]-benzenesulfonamide (11) as a colorless solid (554 mg, 74% yield).

¹H-NMR (CDCl₃): δ 8.15 (d, 1H), 8.10 (s, 1H), 8.03 (s, 1H), 7.68 (d, 1H), 7.55 (t, 1H), 7.46 (t, 1H), 7.27 (d, 1H), 6.47 (d, 1H), 4.91 (s, 2H), 4.84 (t, 1H), 4.44 (d, 2H), 3.76 (s, 1H), 2.40 (s, 3H), 1.55 (s, 6H), 1.03 (s, 9H).

Example 12 Synthesis of N-tert-Butyl-2-[6-(5-hydroxy-4-hydroxymethyl-6-methyl-pyridin-3-ylamino)-pyridin-3-yl]-benzenesulfonamide (12)

The hydrolysis of N-tert-butyl-2-[6-(2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyrdin-5-ylamino)-pyridin-3-yl]-benzenesulfonamide (12) (305 mg, 0.63 mmol), following the procedure described in Example 2, gave N-tert-butyl-2-[6-(5-hydroxy-4-hydroxymethyl-6-methyl-pyridin-3-ylamino)-pyridin-3-yl]-benzenesulfonamide (12) (244 mg, 84% yield) as a colorless solid.

¹H-NMR (CD₃OD): δ 8.13-8.10 (m, 3H), 7.97 (d, 2H), 7.64-7.60 (m, 2H), 7.52 (t, 1H), 7.34 (d, 1H), 6.69 (d, 1H), 5.02 (s, 2H), 4.59 (s, 2H), 2.46 (s, 3H), 1.06 (s, 9H).

Example 13 Synthesis of 4′-{[5-(3-Cyano-benzyloxy)-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl]-amino}-biphenyl-2-sulfonic acid tert-butylamide (13)

A mixture of 4′-[(5-hydroxy-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl)-amino]-biphenyl-2-sulfonic acid tert-butylamide (2) (190 mg, 0.42 mmol), α-bromo-m-tolunitrile (90 mg, 0.46 mmol) and potassium carbonate (177 mg, 1.28 mmol) in DMF (10 mL) were stirred at room temperature under nitrogen atmosphere overnight. The reaction mixture was evaporated to dryness, and the crude product was purified by column chromatography on silica gel using a gradient of dichloromethane:methyl alcohol (1:0 to 9:1) as eluant to give 4′-{[5-(3-cyano-benzyloxy)-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl]-amino}-biphenyl-2-sulfonic acid tert-butylamide (13) (149 mg, 62% yield) as a colorless solid.

¹H-NMR (CDCl₃): δ 8.39 (s, 1H), 8.16 (d, 1H), 7.83 (s, 1H), 7.75-7.68 (m, 2H), 7.59-7.53 (m, 2H), 7.49-7.41 (m, 3H), 7.31 (d, 2H), 6.87 (d, 2H), 5.03 (s, 2H), 4.81 (s, 2H), 4.46 (s, 2H), 3.70 (s, 1H), 2.58 (s, 3H), 1.02 (s, 9H).

Example 14 Synthesis of 3-{4-Hydroxymethyl-5-[(2′-sulfamoyl-biphenyl-4-ylamino)-methyl]-pyridin-3-yloxymethyl}-benzamidine (14)

Hydrogen chloride gas was bubbled into a suspension of 4′-{[5-(3-cyano-benzyloxy)-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl]-amino}-biphenyl-2-sulfonic acid tert-butylamide (13) (100 mg, 0.17 mmol) in absolute ethyl alcohol (30 mL) at 0° C. for 30 min. The septum was replaced and the reaction mixture was stirred at room temperature overnight. Hydrogen chloride gas was purged with nitrogen gas for 2 h and the solvent evaporated to give the crude amide ester as a solid. Ammonia in methyl alcohol (30 mL, 7 M, 350 mmol) was added to the crude amide ester and the reaction mixture was stirred overnight at room temperature. The solvent was evaporated and the product purified on a silica gel column using a mixture of dichloromethane:methyl alcohol (4:1) as eluant to give the corresponding 3-{4-hydroxymethyl-5-[(2′-sulfamoyl-biphenyl-4-ylamino)-methyl]-pyridin-3-yloxymethyl}-benzamidine (14) (90 mg, 97% yield) as a colorless powder.

¹H-NMR (CD₃OD): δ 8.30 (s, 1H), 8.07 (d, 1H), 8.01 (s, 1H), 7.92 (d, 1H), 7.84 (d, 1H), 7.70 (t, 1H) 7.58 (t, 1H), 7.46 (t, 1H), 7.32 (d, 1H), 7.24 (d, 2H), 6.77 (d, 2H), 5.11 (s, 2H), 4.58 (s, 2H), 3.36 (s, 2H), 2.52 (s, 3H). MS m/z (ES⁺): 532.37 (M+H⁺).

Example 15 Synthesis of N-tert-Butyl-2-{6-[3-(3-cyano-benzyloxy)-2-hydroxymethyl-4-methyl-benzylamino]-pyridin-3-yl}-benzenesulfonamide (15)

The coupling of N-tert-butyl-2-[6-(5-hydroxy-4-hydroxymethyl-6-methyl-pyridin-3-ylamino)-pyridin-3-yl]-benzenesulfonamide (12) (205 mg, 0.45 mmol) and α-bromo-m-tolunitrile (88 mg, 0.45 mmol), following the procedure described in Example 13, gave N-tert-butyl-2-{6-[3-(3-cyano-benzyloxy)-2-hydroxymethyl-4-methyl-benzylamino]-pyridin-3-yl}3-benzenesulfonamide (15) (23 mg, 9% yield).

¹H-NMR (CDCl₃): δ 8.38 (s, 1H), 8.14 (dd, 1H), 7.98 (d, 1H), 7.81 (s, 1H), 7.74-7.64 (m, 3H), 7.55-7.46 (m, 3H), 7.24 (dd, 1H), 6.56 (d, 1H), 5.35 (t, 1H), 4.99 (s, 2H), 4.85 (s, 2H), 4.71 (d, 2H), 3.63 (s, 1H), 2.51 (s, 3H), 1.00 (s, 9H).

Example 16 Synthesis of 3-(2-Hydroxymethyl-6-methyl-3-{[5-(2-sulfamoyl-phenyl)-pyridin-2-ylamino]-methyl}-phenoxymethyl)-benzamidine (16)

The conversion of nitrile (15) to amidine (16) was carried out as described in Example 14.

¹H-NMR (CD₃OD): δ 8.32 (s, 1H), 8.11 (dd, 1H), 8.01 (t, 1H), 7.96 (d, 1H), 7.92 (d, 1H), 7.83 (d, 1H), 7.69 (t, 1H), 7.64-7.50 (m, 3H), 7.32 (dd, 1H), 6.67 (d, 1H), 5.12 (s, 2H), 4.88 (s, 2H), 4.73 (s, 2H), 2.52 (s, 3H). MS m/z (ES⁺): 533.42 (M+H⁺).

Example 17 Synthesis of 4′-{[5-(3-Cyano-benzyloxy)-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl]-amino}-biphenyl-2-sulfonic acid tert-butylamide (17)

The coupling of 4′-[(5-hydroxy-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl)-amino]-biphenyl-2-sulfonic acid tert-butylamide (2) (190 mg, 0.42 mmol) and α-bromo-p-tolunitrile (90 mg, 0.46 mmol), following the procedure described in Example 13, gave 4′-{[5-(3-cyano-benzyloxy)-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl]-amino}-biphenyl-2-sulfonic acid tert-butylamide (58) (149 mg, 62% yield) as a colorless solid.

¹H-NMR (CDCl₃): δ 8.39 (s, 1H), 8.16 (d, 1H), 7.83 (s, 1H), 7.75-7.68 (m, 2H), 7.59-7.53 (m, 2H), 7.49-7.41 (m, 3H), 7.31 (d, 2H), 6.87 (d, 2H), 5.03 (s, 2H), 4.81 (s, 2H), 4.46 (s, 2H), 3.70 (s, 1H), 2.58 (s, 3H), 1.02 (s, 9H).

Example 18 Synthesis of 3-{4-Hydroxymethyl-5-[(2′-sulfamoyl-biphenyl-4-ylamino)-methyl]-pyridin-3-yloxymethyl}-benzamidine (18)

The conversion of nitrile (17) to amidine (18) was carried out as described in Example 14.

¹H-NMR (CD₃OD): δ 8.30 (s, 1H), 8.07 (d, 1H), 8.01 (s, 1H), 7.92 (d, 1H), 7.84 (d, 1H), 7.70 (t, 1H) 7.58 (t, 1H), 7.46 (t, 1H), 7.32 (d, 1H), 7.24 (d, 2H), 6.77 (d, 2H), 5.11 (s, 2H), 4.58 (s, 2H), 3.36 (s, 2H), 2.52 (s, 3H). MS m/z (ES⁺): 532.37 (M+H⁺).

Example 19 Synthesis of 3′-Amino-biphenyl-2-sulfonic acid tert-butylamide (19)

Compound (19) was prepared according to the procedure described in Example 11 from 3-bromoanaline (2.08 g, 12.1 mmol) and 2-tert-butylsulfamoyl-phenylboronic acid (2.83 g, 11 mmol). The crude mixture was purified by column chromatography over silica gel using a hexane ethyl acetate (1:1) mixture as eluant to give 2.6 g (84%) of (19).

¹H-NMR (CDCl₃): δ 8.15 (dd, 1H), 7.54 (dt, 1H), 7.45 (dt, 1H), 7.33 (dd, 1H), 7.23 (t, 1H), 6.88 (s, 1H), 6.85 (d, 1H), 6.74 (dd, 1H), 3.76 (s, 1H), 3.40 (br s, 2H), 1.0 (s, 9H).

Example 20 Synthesis of 3′-[(2,2,8-Trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-ylmethyl)-amino]-biphenyl-2-sulfonic acid tert-butylamide (20)

Compound (20) was prepared according to the procedure described in Example 1 from biphenyl amine (19) (913 mg, 3 mmol) and 2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridine-5-carbaldehyde (622 mg, 3 mmol). The crude mixture was purified by column chromatography over silica gel using a hexane/ethyl acetate (1:1) mixture as eluant to give 167 mg (11%) of (20).

¹H-NMR (CDCl₃): δ 8.13 (dd, 1H), 8.02 (s, 1H), 7.55 (dt, 1H), 7.42 (dt, 1H), 7.33 (dd, 1H), 7.26 (t, 1H), 6.90 (s, 1H), 6.82 (dd, 1H), 6.68 (dd, 1H), 4.88 (s, 2H), 4.20 (s, 2H), 3.96 (br s, 1H), 3.66 (s, 1H), 2.40 (s, 3H), 1.54 (s, 6H), 0.97 (s, 9H).

Example 21 Synthesis of 3′-[(5-Hydroxy-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl)-amino]-biphenyl-2-sulfonic acid amide (21)

Compound (20) (105 mg, 0.21 mmol) was dissolved in anhydrous methanol (30 ml), and the solution was cooled to 5° C. then saturated with HCl gas. The solution was allowed to slowly warm to room temperature and stirred overnight. The remaining HCl was removed with a steady stream of nitrogen, and the trace amounts of remaining acid neutralized with 7 N methanolic ammonia. The solvents were removed and the residue purified by column chromatography over silica gel using a dichloromethane/methanol (9:1) mixture as eluant to furnish (21) (52 mg, 62%) as a white powder.

¹H-NMR (CD₃OD): δ 8.08 (dd, 1H), 7.89 (s, 1H), 7.60 (dt, 1H), 7.50 (dt, 1H), 7.34 (dd, 1H), 7.21 (dt, 1H), 6.76-6.70 (m, 3H), 4.96 (s, 2H), 4.33 (s, 2H), 2.41 (s, 3H). MS m/z (ES⁺): 400.26 (M+H⁺).

Example 22 Synthesis of 2′-tert-Butylsulfamoyl-biphenyl-4-carboxylic acid [5-(3-cyano-benzyloxy)-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl]-amide (22)

Compound (22) was prepared according to the procedure described in Example 13 from (5) (156 mg, 0.32 mmol) and α-bromo-m-tolunitrile (69 mg, 0.35 mmol). The crude mixture was purified by column chromatography over silica gel using a mixture of dichloromethane/methyl alcohol (gradient from 1:0 to 9:1) as eluant gave 100 mg (52%) of (22).

¹H-NMR (CD₃OD): δ 8.49 (br s, 1H), 8.12 (dd, 1H), 7.94-7.91 (m, 3H), 7.85 (d, 1H), 7.78 (dd, 1H), 7.63-7.61 (m, 2H), 7.6-7.53 (m, 3H), 7.32 (dd, 1H), 5.18 (s, 2H), 4.96 (s, 2H), 4.90 (s, 2H), 2.7 (s, 3H), 1.06 (s, 9H).

Example 23 Synthesis of 2′-tert-Butylsulfamoyl-biphenyl-4-carboxilic acid[5-(carbamimidoyl-benzyloxy)-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl]-amide (23)

Compound (23) was prepared according to the procedure described in Example 14 from (22) (100 mg, 0.17 mmol). The crude mixture was purified by reverse phase preparatory HPLC (Waters XTerra® Prep RP₁₈ OBD™ (10 μM), 19×250 mm)) using 10% to 100% acetonitrile versus 0.1% aqueous trifluoroacetic acid to provide 30 mg, (32%) of (23).

¹H-NMR (CD₃OD): δ 8.44 (s, 1H), 8.12 (dd, 1H), 7.99 (br s, 1H), 7.93-7.89 (m, 3H), 7.84 (dd, 1H), 7.70 (t, 1H), 7.63 (dd, 1H), 7.58 (dd, 1H), 7.55-7.52 (m, 2H), 7.34 (dd, 1H), 5.20 (s, 2H), 4.95 (s, 2H), 4.83 (s, 2H), 2.52 (s, 3H). MS m/z (ES⁺): 560.57 (M+H⁺).

Example 24 Synthesis of 4′-{[3-(3-Cyano-benzyloxy)-5-hydroxymethyl-2-methyl-pyridin-4-ylmethyl]-amino}-biphenyl-2-sulfonic acid tert-butylamide (24)

Compound (24) was prepared according to the procedure described in Example 13 from (7) (85 mg, 0.19 mmol) and α-bromo-m-tolunitrile (40 mg, 0.2 mmol). The crude mixture was purified by column chromatography over silica gel using a dichloromethane/methyl alcohol (9:1) mixture as eluant to give 62 mg (58%) of (24).

¹H-NMR (CDCl₃): δ 8.33 (br s, 1H), 8.13 (dd, 1H), 7.72 (br s, 1H), 7.64 (m, 2H), 7.52 (m, 2H), 7.45 (dd, 1H), 7.38-7.35 (m, 2H), 7.32 (dd, 1H), 6.76 (m, 2H), 4.96 (s, 2H), 4.79 (s, 2H), 4.42 (s, 2H), 3.7 (s, 1H), 2.60 (s, 3H), 1.02 (s, 9H).

Example 25 Synthesis of 3-{5-Hydroxymethyl-2-methyl-4-[(2′-sulfamoyl-biphenyl-4-ylamino)-methyl]-pyridin-3-yloxymethyl}-benzamidine (25)

Compound (25) was prepared according to the procedure described in Example 14 from (24) (62 mg, 0.11 mmol). The crude mixture was purified by column chromatography over silica gel using a dichloromethane/methanol (gradient from 19:1 to 4:1) mixture as eluant to give 5 mg, (9%) of (25). The major product (33 mg, 56%) was the corresponding benzimidic acid methyl ester (MS m/z (ES⁺): 547.42 (M+H⁺)) as determined by MS.

¹H-NMR (CD₃OD): δ 8.12 (s, 1H), 7.88 (dd, 1H), 7.75 (br s, 1H), 7.58 (dd, 1H), 7.55 (dd, 1H), 7.44-7.37 (m, 2H), 7.29 (dt, 1H), 7.12 (dd, 1H), 7.04 (d, 2H), 6.55 (d, 2H), 4.92 (s, 2H), 4.58 (s, 2H), 4.21 (s, 2H), 2.4 (s, 3H). MS m/z (ES⁺): 532.43 (M+H⁺).

Example 26 Synthesis of 3′-[(3-Hydroxy-5-hydroxymethyl-2-methyl-pyridin-4-ylmethyl)-amino]-biphenyl-2-sulfonic acid tert-butylamide (26)

Compound (26) was prepared according to the procedure described in Example 17 from (19) (1 g, 3.3 mmol) and pyridoxal hydrochloride (802 mg, 3.9 mmol). The crude product was purified by column chromatography over silica gel using a dichloromethane/methanol (9:1) mixture as eluant to give 389 mg (26%) of (26).

¹H-NMR (CDCl₃): δ 8.15 (dd, 1H), 7.88 (s, 1H), 7.53 (t, 1H), 7.49 (t, 1H), 7.35-7.27 (m, 2H), 7.1 (s, 1H), 6.98 (d, 1H), 6.88 (d, 1H), 4.65 (s, 2H), 4.61 (s, 2H), 2.42 (s, 3H), 1.01 (s, 9H).

Example 27 Synthesis of 3′-{[3-(4-Cyano-benzyloxy)-5-hydroxymethyl-2-methyl-pyridin-4-ylmethyl]-amino}-biphenyl-2-sulfonic acid tert-butylamide (27)

Compound (27) was prepared according to the procedure described in Example 13 from (7) (184 mg, 0.4 mmol) and α-bromo-p-tolunitrile (87 mg, 0.44 mmol). The crude mixture was purified by column chromatography over silica gel using a dichloromethane/methyl alcohol (9:1) mixture to furnish 118 mg (51%) of (27).

¹H-NMR (CDCl₃): δ 8.27 (s, 1H), 8.14 (dd, 1H), 7.63 (d, 2H), 7.59-7.47 (m, 2H), 7.53 (d, 2H), 7.3 (dd, 1H), 7.23 (d, 1H), 7.0 (m, 1H), 6.79 (d, 1H), 6.7 (dd, 1H), 4.96 (s, 2H), 4.73 (s, 2H), 4.43 (s, 2H), 3.74 (s, 1H), 2.54 (s, 3H), 0.95 (s, 9H).

Example 28 Synthesis of 4-{5-Hydroxymethyl-2-methyl-4-[(2′-sulfamoyl-biphenyl-3-ylamino)-methyl]-pyridin-3-yloxymethyl}-benzamidine (28)

Compound (28) was prepared according to the procedure described in Example 14 from (27) (103 mg, 0.18 mmol). The crude mixture was purified by column chromatography over silica gel using a dichloromethane/methanol (gradient from 9:1 to 1:1) mixture as eluant to provide 54 mg (56%) of (28).

¹H-NMR (CD₃OD): δ 8.28 (s, 1H), 8.08 (dd, 1H), 7.77 (d, 2H), 7.66 (d, 2H), 7.56 (dt, 1H), 7.5 (dt, 1H), 7.29 (dd, 1H), 7.15 (t, 1H), 6.79 (t, 1H), 6.72-6.69 (m, 2H), 5.08 (s, 2H), 4.76 (s, 2H), 4.37 (s, 2H), 2.53 (s, 3H). MS m/z (ES⁺): 532.37 (M+H⁺).

Example 29 Synthesis of 4′-{[3-(4-Cyano-benzyloxy)-5-hydroxymethyl-2-methyl-pyridin-4-ylmethyl]-amino}-3′-fluoro-biphenyl-2-sulfonic acid tert-butylamide (29)

Compound (29) was prepared according to the procedure described in Example 13 from (9) (137 mg, 0.29 mmol) and α-bromo-p-tolunitrile (62 mg, 0.45 mmol). The crude mixture was purified by column chromatography over silica gel using a dichloromethane/methyl alcohol (19:1) mixture as eluant to give 94 mg (55%) of (29).

¹H-NMR (CD₃OD): δ 8.28 (s, 1H), 8.07 (dd, 1H), 7.97 (br s, 1H), 7.71 (d, 2H), 7.63 (d, 2H), 7.58 (dt, 1H), 7.47 (dt, 1H), 7.32 (dd, 1H), 7.12 (dd, 1H), 7.04 (dd, 1H), 6.87 (t, 1H), 5.07 (s, 2H), 4.77 (s, 2H), 4,47 (s, 2H), 2.54 (s, 3H), 0.98 (s, 9H).

¹⁹F-NMR (CD₃OD; ¹H-decoupled): δ −137.9.

Example 30 Synthesis of 4-{4-[(3-Fluoro-2′-sulfamoyl-biphenyl-4-ylamino)-methyl]-5-hydroxymethyl-2-methyl-pyridin-3-yloxymethyl}-benzamidine (30)

Compound (30) was prepared according to the procedure described in Example 14 from (29) (65 mg, 0.11 mmol). The crude mixture was purified by column chromatography over silica gel using a dichloromethane/methanol (9:1) mixture as eluant to give 41 mg (84%) of (30).

¹H-NMR (MeOH-d₄): δ 8.29 (s, 1H), 8.07 (dd, 1H), 7.81 (d, 2H), 7.69 (d, 1H), 7.58 (dt, 1H), 7.49 (dt, 1H), 7.3 (dd, 1H), 7.08 (dd, 1H), 7.04 (dd, 1H), 6.85 (t, 1H), 5.12 (s, 2H), 4.78 (s, 2H), 4.47 (s, 2H), 2.57 (s, 3H).

¹⁹F-NMR (CD₃OD; ¹H-decoupled): 6-137.8. MS m/z (ES⁺): 550.56 (M+H⁺).

Example 31 Synthesis of 2-{6-[(5-Hydroxy-4-hydroxymethyl-6-methyl-pyridin-3-ylmethyl)-amino]-pyridin-3-yl}-benzenesulfonamide (31)

Compound (31) was prepared from (12) (90 mg, 0.2 mmol) following the procedure described in Example 3. The crude mixture was purified by column chromatography over silica gel using a dichloromethane/methanol (9:1) mixture as eluant to furnish 19 mg (24%) of (31).

¹H-NMR (CD₃OD): δ 7.94 (d, 1H), 7.89 (s, 1H), 7.81 (s, 1H), 7.45 (m, 4H), 7.22 (dd, 1H), 6.51 (d, 1H), 4.87 (s, 2H), 4.44 (s, 2H), 2.31 (s, 3H).

Example 32 Synthesis of 3′-[(3-Hydroxy-5-hydroxymethyl-2-methyl-pyridin-4-ylmethyl)-amino]-biphenyl-2-sulfoniamide (32)

This compound was prepared from compound (26) (100 mg, 0.22 mmol) according to the procedure described for Example 3. The crude mixture was purified by column chromatography over silica gel using a dichloromethane/methanol (9:1) mixture as eluant. to provide 80 mg, (91%) of (32).

¹H-NMR (CD₃OD): δ 8.08 (dd, 1H), 7.86 (s, 1H), 7.59 (dt, 1H), 7.5 (dt, 1H), 7.32 (dd), 7.24 (dt, 1H), 6.88-6.83 (m, 3H), 4.70 (s, 2H), 4.53 (s, 2H), 2.4 (s, 2H).

Example 33 Inhibition of Platelet Aggregation

Platelet rich plasma (PRP) was obtained by drawing whole blood from normal human donors (not on any medication) into sodium citrate tubes (3.2%), and centrifuging at 160×g for about 10 minutes. Platelet poor plasma (PPP) was obtained by centrifuging the remainder of the sample after the platelets were removed at 800×g for about 10 minutes. The PRP was adjusted to a count of 280×10⁹/L using a mixture of PRP and PPP. The platelets (200 μL) were incubated with the test compounds (25 μL) adjusted to various concentrations (from a 250 μM stock solution) for about 30 minutes at room temperature (approximate final platelet count in the incubation mixture of 250×10⁹/L). The samples were incubated for about 3 minutes at about 37° C., and then transferred to the mixing wells of a Chrono-log 4 channel aggregometer (Chrono-log Corp., Havertown, Pa.). After baselines were established, the agonist (25 μL of 40 μM ADP (Sigma, St. Louis, Mo.) or 25 μL of 50 μg/mL and 10 μg/mL collagen (Helena Laboratories, Beaumont, Tex.) or 25 μL of 120 μM thrombin receptor activating peptide (TRAP) (Sigma)) was then added. Aggregation was monitored for 5 minutes at 37° C. with stirring (1000 rpm). The amplitude and slope of each tracing were calculated to determine the amount of aggregation. Control samples were performed using only solvent. The % reduction in aggregation was calculated for each sample compared to the proper solvent control. See Table 1. TABLE 1 Platelet inhibition % Reduction in Aggregation Concentration Collagen Collagen ADP TRAP Compound (μM) (5 μg/mL) (1 μg/mL) (4 μM) (12 μM) 3 250 5 0 0 6 6 250 0 5 0 1 14 250 5 15 10 9 16 250 0 14 3 1.3 18 250 9 23 50 74 21 250 0 4 14 0 28 250 15 10 26 11 30 100 19 57 23 57 33 500 15 5 4 1 34 250 1 41 63 93 Compound 33 is Compound XXXV in U.S. Pat. No. 6,417,204 B1, which is hereby incorporated by reference, and compound 34 is Compound XL in U.S. Pat. No. 6,417,204 B1

Example 34 Inhibition of Serine Proteases

Compounds were evaluated for inhibition of serine protease activity.

Methods

The enzymes factor Xa (fXa), thrombin (factor IIa or fIIa), and trypsin were purchased from Haematologic Technologies Inc. (Essex Junction, Vt.). The substrates Pefachromes Xa, Th, and Try were purchased from Centerchem Inc. (Stamford, Conn.). Varying concentrations of the sulfonamide compounds were incubated for 30 min at room temperature with purified activated human enzyme in the appropriate buffer. Various concentrations of the corresponding synthetic substrate were added, and activity was measured at 37° C. by monitoring absorbance at 405 nm for 15 min with a Flurostar Optima plate reader. Each assay had a final volume of 200 μL, and assays were performed in 96-well plates. For IC₅₀ determinations, 300 μM of substrate was tested against five concentrations (50, 100, 200, 300, and 500 μM) of test compound plus control (run in duplicate). For controls, the appropriate percentage of solvent was used instead of test compound. The specific activity (ΔA₄₀₅/min/nM protein), and the percent activity was calculated for each data point. The IC₅₀ was calculated by linear regression of log[test compound] vs. % activity. The enzyme activity of the sulfonamide compound was determined using the Enzyme Kinetics Module of Sigmaplot 8.0 (SPSS Inc.)

The final concentration of human Factor Xa was 2 nM in a Tris/NaCl buffer (pH 7.5) containing 0.25% (w/v) polyethylene glycol (MW 8000). The substrate employed was Pefachrome Xa.

The final concentration of human thrombin was 2 nM in a Tris/NaCl buffer (pH 8.3) containing 0.125% (w/v) bovine serum albumin. The substrate used was Pefachrome Th.

The final concentration of human trypsin was 2 nM in a Tris/NaCl buffer (pH 7.5) containing 0.25% (w/v) bovine serum albumin and 20 μM CaCl₂. The substrate used was Pefachrome Try.

The serine protease activity (fXa, fIIa and trypsin) of the pyridoxine sulfonamides (Table 2) showed that some of these compounds may serve as compounds with improved anticoagulant (possibly anti-fxa) properties. Compounds 3, 8, 31, and 32 were virtually inactive against all three serine proteases (>1000 μM). Unexpectedly, the N-blocked sulfonamides 2, 7, and 12 displayed some enhanced, albeit low, inhibition of fXa activity over their deprotected counterparts 3, 8, and 31, respectively. An exception to this was noted in the case where the linker contained an amide as exemplified by compounds 5 and 6, where both the N-protected and free sulfonamide showed similar fXa activity. As could be expected, significant improvement in inhibition of flia and especially fXa activity was observed upon incorporation of the basic benzamidine moiety that is anticipated to interact with Asp 189 in the S1 pocket. For the pyridoxine scaffold described herein, the 4-position biaryl isomer (compound 25) displayed similar overall inhibitory properties when compared to the corresponding 5-position biaryl isomers (e.g., compound 14), with a slight improvement in inhibition of fXa activity observed with the former. However, neither of these arrangements showed significant improvements in terms of selectivity over trypsin. Also, insertion of a carbonyl improved inhibition of fIIa activity (see 23 versus 14). TABLE 2 Inhibition of serine protease (fXa, fIIa and Trypsin) activity by sulfonamides Compound IC₅₀ (fXa) (μM) IC₅₀ (fIIa) (μM) IC₅₀ (Trypsin) (μM) 2 329 >1000 >1000 3 >1000 >1000 >1000 5 362 >1000 >1000 6 306 >1000 >1000 7 197 >1000 >1000 8 >1000 >1000 >1000 12 317 >1000 >1000 14 15 862 12 16 11 >1000 11 18 32 370 43 21 326 >1000 >1000 23 5 97 24 25 4 >1000 15 28 17 476 3 30 18 171 4 31 >1000 >1000 >1000 32 >1000 >1000 >1000 33 105 300 18 34 29 >1000 4 

1. A method of inhibiting serine protease activity comprising: administering a compound of the formula:

wherein R¹ is —OH, —O-alkyl, —(CH₂)_(n′)OH where n′ is an integer from 1 to 8, alkyl, cycloalkyl, or O-alkyl-aryl-R⁴, where R⁴ is —CN or amidine; R² is alkyl; —(CH₂)_(n′)OH where n′ is as defined above; —(CH₂)_(n)COOH where n is an integer from 0 to 8; —(CH₂)_(n)COO(CH₂)_(n)CH₃ where n is as defined above; or R⁵ where R⁵ is (CH₂)_(n)-aryl-R⁶ where n is as defined above and R⁶ is SO₂NH₂ or SO₂NHC(CH₃)₃; (CH₂)_(n)-aryl-aryl-R⁶, where n and R⁶ are as defined above; (CH₂)_(n)—NH-aryl-aryl-R⁶, where n and R⁶ are as defined above; (CH₂)_(n)—NH—CO-aryl-aryl-R⁶, where n and R⁶ are as defined above; or —(CH₂)_(n)—NH-aryl-R⁶, where n and R⁶ are as defined above; R³ is alkyl; —(CH₂)_(n′)OH where n′ is as defined above; or R⁵ where R⁵ is (CH₂)_(n)—NH-aryl-R⁶, where n and R⁶ are as defined above; (CH₂)_(n)—NH—CO-aryl-R where n and R⁶ are as defined above; (CH₂)_(n)—NH-aryl-aryl-R⁶ where n and R⁶ are as defined above; or (CH₂)_(n)—NH—CO-aryl-aryl-R⁶ where n and R⁶ are as defined above; further wherein R² is alkyl or —(CH₂)_(n′)OH where n′ is as defined above, and R³ is R⁵; or R² is R⁵ and R³ is alkyl or —(CH₂)_(n′)OH where n′ is as defined above; and R¹ and R² when taken together form compounds of formula II,

wherein R⁷ and R⁸ are independently H or CH₃, and R⁵ is as defined above; or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1, wherein said serine protease is Factor Xa.
 3. The method of claim 1, wherein said serine protease is Factor IIa.
 4. The method of claim 1, wherein said serine protease is trypsin.
 5. The method of claim 1, wherein at least one alkyl is substituted with one or more of fluorine, chlorine, alkoxy groups having 1 to 8 carbon atoms, or amido groups having from 1 to 8 carbon atoms.
 6. The method of claim 5, wherein the alkoxy group is methoxy or ethoxy.
 7. The method of claim 5, wherein the amido group is acetamido.
 8. The method of claim 1, wherein the aryl group is a phenyl group or a naphthyl group.
 9. The method of claim 1, wherein the aryl group is substituted with one or more of fluorine, chlorine, bromine, alkyl groups having 1 to 8 carbon atoms, alkoxy groups having 1 to 8 carbon atoms, alkoxyalkyl groups having 1 to 8 carbon atoms, or amido groups having 1 to 8 carbon atoms.
 10. The method of claim 9, wherein the alkyl group is methyl or ethyl.
 11. The method of claim 9, wherein the alkoxy group is methoxy or ethoxy.
 12. The method of claim 9, wherein the amido group is acetamido.
 13. The method of claim 1, wherein the aryl group is substituted with one or more functional groups.
 14. The method of claim 13, wherein the functional group is a hydroxy group, carboxy group, or acetoxy group.
 15. A method of inhibiting serine protease activity comprising: administering a compound of the formula

wherein R₁ is OH; OCH₃; OCH₂-(4-tert-Butyl-phenyl); or

where R⁴ is —CN or amidine; R² is (CH₂)_(m)OH, where m=0 to 8; or R⁵, where R⁵ is

 where W is (CH₂)_(n) where n=1, 2, or 3; where X is C=0 or (CH₂)_(n′), where n′=0, 1, 2, or 3; Y is CH, CF, or N; and R⁶ is

R³ is (CH₂)_(m)OH, where m is as defined above; or R⁵, where R⁵ is as defined above; further wherein R² is (CH₂)_(m)OH, where m is as defined above, and R³ is R⁵; or R² is R⁵ and R³ is (CH₂)_(m)OH, where m is as defined above; and R¹ and R² when taken together form a compound of formula IV

wherein R⁷ and R⁸ are independently H or CH₃; and R⁵ is as defined above; or a pharmaceutically acceptable salt thereof.
 16. The method of claim 15, wherein said serine protease is Factor Xa.
 17. The method of claim 15, wherein said serine protease is Factor Ia.
 18. The method of claim 15, wherein said serine protease is trypsin.
 19. The method of claim 15, wherein said compound is administered enterally, parenterally, or by inhalation.
 20. The method of claim 15, wherein the compound is administered concurrently with another therapeutic agent.
 21. The method of claim 20, wherein said other therapeutic agent is an anti-platelet agent, glycoprotein IIb/IIIa inhibitor, or anticoagulant.
 22. The method of claim 21, wherein said anti-platelet agent is clopidogrel, aspirin, or dipyridamole.
 23. The method of claim 21, wherein said glycoprotein IIb/IIIa inhibitor is eptifibatide.
 24. The method of claim 21, wherein said anticoagulant is unfractionated heparin, low molecular weight heparin, hirudin, or argatroban.
 25. A method of inhibiting in vitro serine protease activity comprising: contacting an isolated cell with a compound of the formula:

wherein R¹ is —OH, —O-alkyl, —(CH₂)_(n′)OH where n′ is an integer from 1 to 8, alkyl, cycloalkyl, or O-alkyl-aryl-R⁴, where R⁴ is —CN or amidine; R² is alkyl; —(CH₂)_(n′)OH where n′ is as defined above; —(CH₂)_(n)COOH where n is an integer from 0 to 8; —(CH₂)_(n)COO(CH₂)_(n)CH₃ where n is as defined above; or R⁵ where R⁵ is (CH₂)_(n)-aryl-R⁶ where n is as defined above and R⁶ is SO₂NH₂ or SO₂NHC(CH₃)₃; (CH₂)_(n)-aryl-aryl-R⁶, where n and R⁶ are as defined above; (CH₂)_(n)—NH-aryl-aryl-R⁶, where n and R⁶ are as defined above; (CH₂)_(n)—NH—CO-aryl-aryl-R⁶, where n and R⁶ are as defined above; or —(CH₂)_(n)—NH-aryl-R⁶, where n and R⁶ are as defined above; R³ is alkyl; —(CH₂)_(n′)OH where n′ is as defined above; or R⁵ where R⁵ is (CH₂)_(n)—NH-aryl-R⁶, where n and R⁶ are as defined above; (CH₂)_(n)—NH—CO-aryl-R⁶ where n and R⁶ are as defined above; (CH₂)_(n)—NH-aryl-aryl-R⁶ where n and R⁶ are as defined above; or (CH₂)_(n)—NH—CO-aryl-aryl-R⁶ where n and R⁶ are as defined above; further wherein R² is alkyl or —(CH₂)_(n′)OH where n′ is as defined above, and R³ is R⁵; or R² is R⁵ and R³ is alkyl or —(CH₂)_(n′)OH where n′ is as defined above; and R¹ and R² when taken together form compounds of formula II,

wherein R⁷ and R⁸ are independently H or CH₃, and R⁵ is as defined above; or a pharmaceutically acceptable salt thereof.
 26. A method of inhibiting serine protease activity comprising: contacting an organism with a compound of the formula:

wherein R¹ is —OH, —O-alkyl, —(CH₂)_(n′)OH where n′ is an integer from 1 to 8, alkyl, cycloalkyl, or O-alkyl-aryl-R⁴, where R⁴ is —CN or amidine; R² is alkyl; —(CH₂)_(n′)OH where n′ is as defined above; —(CH₂)_(n)COOH where n is an integer from 0 to 8; —(CH₂)_(n)COO(CH₂)_(n)CH₃ where n is as defined above; or R⁵ where R⁵ is (CH₂)_(n)-aryl-R⁶ where n is as defined above and R⁶ is SO₂NH₂ or SO₂NHC(CH₃)₃; (CH₂)_(n)-aryl-aryl-R⁶, where n and R⁶ are as defined above; (CH₂)_(n)—NH-aryl-aryl-R⁶, where n and R⁶ are as defined above; (CH₂)_(n)—NH—CO-aryl-aryl-R⁶, where n and R⁶ are as defined above; or —(CH₂)_(n)—NH-aryl-R⁶, where n and R⁶ are as defined above; R³ is alkyl; —(CH₂)_(n)OH where n′ is as defined above; or R⁵ where R⁵ is (CH₂)_(n)—NH-aryl-R⁶, where n and R⁶ are as defined above; (CH₂)_(n)—NH—CO-aryl-R⁶ where n and R⁶ are as defined above; (CH₂)_(n)—NH-aryl-aryl-R⁶ where n and R⁶ are as defined above; or (CH₂)_(n)—NH—CO-aryl-aryl-R⁶ where n and R⁶ are as defined above; further wherein R² is alkyl or —(CH₂)_(n′)OH where n′ is as defined above, and R³ is R⁵; or R² is R⁵ and R³ is alkyl or —(CH₂)_(n′)OH where n′ is as defined above; and R¹ and R² when taken together form compounds of formula II,

wherein R⁷ and R⁸ are independently H or CH₃, and R⁵ is as defined above; or a pharmaceutically acceptable salt thereof.
 27. A compound of the formula

wherein R¹ is OH; OCH₃; OCH₂-(4-tert-Butyl-phenyl); or

where R⁴ is —CN or amidine; R² is (CH₂)_(m)OH, where m=0 to 8; or R⁵, where R⁵ is

 where W is (CH₂)_(n) where n=1, 2, or 3; where X is C=0 or (CH₂)_(n′), where n′=0, 1, 2, or 3; Y is CH, CF, or N; and R⁶ is

R³ is (CH₂)_(m)OH, where m is as defined above; or R⁵, where R⁵ is as defined above; further wherein R² is (CH₂)_(m)OH, where m is as defined above, and R³ is R⁵; or R² is R⁵ and R³ is (CH₂)_(m)OH, where m is as defined above; and R¹ and R² when taken together form a compound of formula IV

wherein R⁷ and R⁸ are independently H or CH₃; and R⁵ is as defined above; or a pharmaceutically acceptable salt thereof.
 28. A method of treating cardiovascular or related diseases in a mammal comprising administering a therapeutically effective amount of a compound of claim
 27. 29. The method of claim 29, wherein the compound is administered concurrently with another therapeutic agent.
 30. The method of claim 29, wherein said other therapeutic agent is an anti-platelet agent, glycoprotein IIb/IIIa inhibitor, or anticoagulant.
 31. The method of claim 30, wherein said anti-platelet agent is clopidogrel, aspirin, or dipyridamole.
 32. The method of claim 30, wherein said glycoprotein IIb/IIIa inhibitor is eptifibatide.
 33. The method of claim 30, wherein said anticoagulant is unfractionated heparin, low molecular weigh heparins, hirudin, or argatroban. 